Establishment of Efficient Cloning Method for Variable Region Genes of Antigen Specific Human Monoclonal Antibody

Abstract

We have developed an in vitro immunization protocol of human peripheral blood mononuclear cells (PBMC) for generating human antigen-specific antibodies. By using this protocol, B cells producing antigen-specific antibody can be propagated within a week. In the present study, we tried to establish an efficient strategy to clone variable region genes of antigen-specific human monoclonal antibody by applying in vitro immunized PBMC to the phage display method. To evaluate the efficiency of our strategy, variable region genes prepared by PCR from PBMC immunized in vitro with mite-extract (ME) and non-immunized PBMC were applied to the phage display method. After concentrating the ME-specific phage antibody by several rounds of panning, the number of phage antibodies specific for ME and antigen specificity both increased by using in vitro immunized PBMC. These results demonstrate that efficiency in acquisition of variable region genes of antigen specific human monoclonal antibody can be improved by in vitro immunizing PBMC used as a source of antibody genes.